The NLRP3 inflammasome in viral infection (Review).

The interplay between pathogen and host determines the immune response during viral infection. The Nod­like receptor (NLR) protein 3 inflammasome is a multiprotein complex that induces the activation of inflammatory caspases and the release of IL­1ß, which play an important role in the innate immune responses. In the present review, the mechanisms of the NLR family pyrin domain containing 3 inflammasome activation and its dysregulation in viral infection were addressed.

0.5-5% Supramolecular Salicylic Acid Hydrogel is Safe for Long-Term Topical Application and Improves the Expression of Genes Related to Skin Barrier Homeostasis in Mice Models.

Background: As a keratolytic, salicylic acid (SA) can be topically applied in various formulations and doses in dermatology. Supramolecular SA hydrogel, a new SA formulation with higher bioavailability, is developed and commercially available nowadays. However, there still remain concerns that the long-term and continual application of SA at low concentrations may jeopardize the cutaneous barrier properties. Aim of the Study: To reveal the long-term effects of 0.5-5% supramolecular SA hydrogel on the skin barrier in normal mice models. Materials and Methods: The 0.5%, 1%, 2%, and 5% supramolecular SA hydrogel or hydrogel vehicle without SA was applied to mice's shaved dorsal skin once per day respectively. Tissue samples of the dorsal skin were harvested on day 14 and 28 of the serial application of SA for histopathological observation and transcriptomic analysis. Results: Following topical supramolecular SA hydrogel therapy with various concentrations of SA (0.5%, 1%, 2%, and 5%) for 14 days and 28 days, there were no obvious macroscopic signs of impaired cutaneous health and no inflammatory or degenerative abnormalities were observed in histological results. Additionally, the transcriptomic analysis revealed that on day 14, SA dramatically altered the expression of genes related to the extracellular matrix structural constituent. And on day 28, SA regulated gene expression profiles of keratinization, cornified envelope, and lipid metabolism remarkably. Furthermore, the expression of skin barrier related genes was significantly elevated after the application of SA based on RNA-seq results, and this is likely to be associated with the PPAR signaling pathway according to the enrichment analysis. Conclusion: Our findings demonstrated that the sustained topical administration of the 0.5-5% supramolecular SA hydrogel for up to 28 days did no harm to normal murine skin and upregulated the expression of genes related to the epidermal barrier.

Human umbilical cord mesenchymal stem cell treatment alleviates symptoms in an atopic dermatitis-like mouse model.

BACKGROUND: Atopic dermatitis (AD) is one of the most common immune and inflammatory skin disorders, leading to insufferable itching and skin abnormalities that seriously affect life quality of patients. There are still huge unmet needs for long-term and effective disease control, despite currently available therapies. Evidenced by some preclinical and clinical studies of AD treatment with stem cells, stem cell treatment could significantly and effectively ameliorate AD symptoms. OBJECTIVES: To elucidate underlying mechanisms of how stem cells therapy alleviates AD-like symptoms. METHODS: An AD-like mouse model was constructed and treated with mesenchymal stem cells (MSCs) subcutaneously or subcutaneously combined with intravenously. The differentially expressed genes were sorted out from RNA sequencing results of dorsal skin and blood. RESULTS: Two injection routes of MSCs could alleviate AD-like symptoms and pathologic changes of the skin and immune organs. RNA sequencing of dorsal skin sections and blood provided gene expression signatures for amelioration of skin defects, inflammatory and immune modulation by MSCs, as well as common AD molecular markers for the skin and blood, which may benefit for clinical diagnosis. IL-1ß and its signaling pathway were specifically found to be associated with the development of AD-like dermatitis lesions. MSC treatment effectively inhibited the JAK-STAT pathway and receptors of IL-4, IL-13, IL-17, and IgE. CONCLUSIONS: MSC therapy could regulate abnormal immune and inflammatory status in AD. Mechanistic exploration will contribute to the development of personalized AD treatment based on MSCs.

RNA sequencing and metabolic analysis of imiquimod-induced psoriasis-like mice with chronic restrain stress.

AIM: Psoriasis is one of the most common dermatological disorders, characterized by increased epidermal hyperplasia and immune cell infiltration. Psychological stress has been reported to contribute to the severity, aggravation, and relapse of psoriasis. However, the exact mechanism involved in psychological stress's impact on psoriasis is still unclear. We aim to investigate the role of psychological stress in psoriasis from a transcriptomic and metabolomic perspective. MAIN METHOD: We developed a chronic restrain stress (CRS)-imiquimod (IMQ)-induced psoriasis-like mouse model and performed a comprehensive comparative transcriptomic and metabolic analysis with control mice, CRS-treated mice, and IMQ-treated mice to investigate how psychological stress affects psoriasis. KEY FINDING: We found that CRS-IMQ-induced psoriasis-like mice showed significant exacerbation of psoriasis-like skin inflammation compared with mice treated with IMQ only. Mice of the CRS + IMQ group showed increased expression of keratinocyte proliferation and differentiation genes, differential regulation of cytokines, and promotion of linoleic acid metabolism. Correlation analysis of differentially expressed genes in the CRS-IMQ-induced psoriasis-like mice and human psoriasis datasets compared with respective controls revealed 96 overlapping genes of which 30 genes showed consistent induced or repressed expression in all human and mouse datasets. SIGNIFICANCE: Our study provides new insights into the effects of psychological stress on psoriasis pathogenesis and the mechanisms involved, which provides clues for development of therapeutics or biomarkers.

γ-Aminobutyric acid promotes the inhibition of hair growth induced by chronic restraint stress.

Stress plays a critical role in hair loss, although the underlying mechanisms are largely unknown. γ-aminobutyric acid (GABA) has been reported to be associated with stress; however, whether it affects stress-induced hair growth inhibition is unclear. This study aimed to investigate the potential roles and mechanisms of action of GABA in chronic restraint stress (CRS)-induced hair growth inhibition. We performed RNA-seq analysis and found that differentially expressed genes (DEGs) associated with neuroactive ligand-receptor interaction, including genes related to GABA receptors, significantly changed after mice were treated with CRS. Targeted metabolomics analysis and enzyme-linked immunosorbent assay (ELISA) also showed that GABA levels in back skin tissues and serum significantly elevated in the CRS group. Notably, CRS-induced hair growth inhibition got aggravated by GABA and alleviated through GABAA antagonists, such as picrotoxin and ginkgolide A. RNA sequencing analysis revealed that DEGs related to the cell cycle, DNA replication, purine metabolism, and pyrimidine metabolism pathways were significantly downregulated in dermal papilla (DP) cells after GABA treatment. Moreover, ginkgolide A, a GABAA antagonist extracted from the leaves of Ginkgo biloba, promoted the cell cycle of DP cells. Therefore, the present study demonstrated that the increase in GABA could promote CRS-induced hair growth inhibition by downregulating the cell cycle of DP cells and suggested that ginkgolide A may be a promising therapeutic drug for hair loss.

Therapeutic potential of mesenchymal stem cells for refractory inflammatory and immune skin diseases.

Inflammatory and immunological skin diseases such as psoriasis, systemic sclerosis, dermatomyositis and atopic dermatitis, whose abnormal skin manifestations not only affected life quality but also caused social discrimination, have been wildly concerned. Complex variables such as hereditary predisposition, racial differences, age and gender can influence the prevalence and therapeutic options. The population of patients with unsatisfactory curative effects under current therapies is growing, it's advisable to seek novel and advanced therapies that are less likely to cause systemic damage. Mesenchymal stem cells (MSCs) have been proven with therapeutic benefits in tissue regeneration, self-renewal and differentiation abilities when treating refractory skin disorders in preclinical and clinical studies. Here we highlighted the immune modulation and inflammation suppression of MSCs in skin diseases, summarized current studies, research progress and related clinical trials, hoping to strengthen the confidence of promising MSCs therapy in future clinical application.

Gene Expression Profile Analysis of Human Epidermal Keratinocytes Expressing Human Papillomavirus Type 8 E7.

Background: Human papillomavirus type 8 (HPV8) has been implicated in the progress of non-melanoma skin cancers and their precursor lesions. The HPV8 E7 oncoprotein plays a key role in the tumorigenesis of HPV-associated cutaneous tumors. However, the exact role of HPV8 E7 in human epidermal carcinogenesis has not been fully elucidated. Methods: To investigate the potential carcinogenic effects of HPV8 E7 on epithelial cells, we used RNA-sequencing technology to analyze the gene expression profile of HPV8 E7-overexpressed normal human epidermal keratinocytes (NHEKs). Results: RNA-sequencing revealed 831 differentially expressed genes (DEGs) between HPV8 E7-expressing NHEKs and control cells, among which, 631 genes were significantly upregulated, and 200 were downregulated. Gene ontology annotation enrichment analysis showed that HPV8 E7 mainly affected the expression of genes associated with protein heterodimerization activity, DNA binding, nucleosomes, and nucleosome assembly. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that overexpression of HPV8 E7 affected the expression of gene clusters associated with viral carcinogenesis and transcriptional misregulation in cancer and necroptosis signaling pathways that reportedly play crucial roles in HPV infection promotion and cancer progression. We also found the DEGs, such as HKDC1 and TNFAIP3, were associated with epigenetic modifications, immune regulation, and metabolic pathways. Conclusion: Our results demonstrate that the pro-carcinogenic effect of HPV8 expression in epithelial cells may be attributed to the regulatory effect of oncogene E7 on gene expression associated with epigenetic modifications and immune and metabolic status-associated gene expression. Although our data are based on an in vitro experiment, it provides the theoretical evidence that the development of squamous cell carcinoma can be caused by HPV.

Human Papillomavirus Type 16 Early Protein E7 Activates Autophagy through Inhibition of Dual-Specificity Phosphatase 5.

Consistent high-risk human papillomavirus (HPV) infection leads to various malignant cancers. Autophagy can promote cancer progression by helping cancer cells survive under stress or induce oncogenic effects when mutations or abnormalities occur. Mitogen activated protein kinases (MAPKs) can transduce various external or intrinsic stimuli into cellular responses, including autophagy, and dual-specificity phosphates (DUSPs) contribute to the direct regulation of MAPK activities. Previously, we showed that expression of DUSP5 was repressed in HPV16 E7-expressing normal human epidermal keratinocytes (NHEKs). Here we show that clinical HPV16 E7-positive precancerous and cancerous tissues also demonstrate low DUSP5 levels compared with control tissues, indicating that the inverse correlation between HPV16 E7 and DUSP5 is clinically relevant. We furthermore investigated the autophagy response in both DUSP5-deficient and HPV16 E7-expressing NHEKs. Confocal microscopy and Western analysis showed induction of LC3-II levels, autophagosome formation and autophagy fluxes in DUSP5-deficient NHEKs. Furthermore, Western analysis demonstrated specific induction of phosphorylated ERK in DUSP5-deficient and HPV16 E7-expressing NHEKs, indicating that HPV16 E7-mediated repression of DUSP5 results in induced MAPK/ERK signaling. Finally, phosphorylated mTOR and ULK (S757) were reduced in DUSP5-deficient NHEKs, while phosphorylated ULK (S555) and AMPK were increased, thereby inducing canonical autophagy through the mTOR and AMPK pathways. In conclusion, our results demonstrate that HPV16 E7 expression reduces DUSP5 levels, which in turn results in active MAPK/ERK signaling and induction of canonical autophagy through mTOR and MAPK regulation. Given its demonstrated inverse correlation with clinical cancerous tissues, DUSP5 may serve as a potential therapeutic target for cervical cancer.

A Novel Combined Use of Dupilumab for Treatment of Aggressive Refractory Pemphigus Vulgaris Complicated With Pulmonary Tuberculosis: A Case Report and the RNA-seq Analysis.

Background: Pemphigus vulgaris (PV) is a kind of IgG-mediated autoimmune blistering disease (AIBD) that is characterized by loss of keratinocyte adhesion in the epithelium of mucous membranes or skin. Recently, pemphigus vulgaris was thought to be associated with classical T helper 2 (TH2)-type cytokines such as interleukin-4 (IL-4) and interleukin-17 (IL-17) signaling pathway. A humanized monoclonal IgG4 antibody called dupilumab binds to the alpha subunit of the interleukin-4 receptor (IL-4Rα) and inhibits the signaling of IL-4 and interleukin-13 (IL-13), which has been successfully applied for atopic dermatitis and asthma. Currently, the clinical trial evaluating dupilumab in bullous pemphigoid is ongoing. Objective: To determine whether dupilumab may be of benefit in the aggressive refractory pemphigus vulgaris. Methods: We report a 35-year old male with refractory pemphigus vulgaris and pulmonary tuberculosis who received treatment with dupilumab for 10 weeks. The mRNA expression of peripheral blood mononuclear cells (PBMCs) was analyzed by RNA sequencing (RNA-seq) which showed the gene expression changes after treatment. Results: The skin lesions of the patient improved in response to the combined use of dupilumab, moderate dose of glucocorticosteroids, and intravenous immune globulin (IVIG). Downregulations of inflammatory response-related genes and IL-17 signaling pathway-related genes were observed in PBMCs. Conclusion: We describe a patient with refractory pemphigus vulgaris and pulmonary tuberculosis who had the disease under control with combined use of dupilumab as an add-on treatment. Dupilumab may provide a beneficial effect in aggressive refractory pemphigus vulgaris.

Integrated Metabolomics and Transcriptomics Analyses Reveal Histidine Metabolism Plays an Important Role in Imiquimod-Induced Psoriasis-like Skin Inflammation.

Psoriasis is a chronic inflammatory skin disease characterized by massive keratinocyte proliferation and immune cell infiltration into the epidermis. However, the specific mechanisms underlying the development of psoriasis remain unclear. Untargeted metabolomics and transcriptomics have been used separately to profile biomarkers and risk genes in the serum of psoriasis patients. However, the integration of metabolomics and transcriptomics to identify dysregulated metabolites and genes in the psoriatic skin is lacking. In this study, we performed an untargeted metabolomics analysis of imiquimod (IMQ)-induced psoriasis-like mice and healthy controls, and found that levels of a total of 4,188 metabolites differed in IMQ-induced psoriasis-like mice compared with those in control mice. Metabolomic data analysis using MetaboAnalyst showed that the metabolic pathways of primary metabolites, such as folate biosynthesis and galactose metabolism, were significantly altered in the skin of mice after treatment with IMQ. Furthermore, IMQ treatment also significantly altered metabolic pathways of secondary metabolites, including histidine metabolism, in mouse skin tissues. The metabolomic results were verified by transcriptomics analysis. RNA-seq results showed that histamine decarboxylase (HDC) mRNA levels were significantly upregulated after IMQ treatment. Targeted inhibition of histamine biosynthesis process using HDC-specific inhibitor, pinocembrin (PINO), significantly alleviated epidermal thickness, downregulated the expression of interleukin (IL)-17A and IL-23, and inhibited the infiltration of immune cells during IMQ-induced psoriasis-like skin inflammation. In conclusion, our study offers a validated and comprehensive understanding of metabolism during the development of psoriasis and demonstrated that PINO could protect against IMQ-induced psoriasis-like skin inflammation.

Metabolomic Analysis of the Ameliorative Effect of Enhanced Proline Metabolism on Hypoxia-Induced Injury in Cardiomyocytes.

BACKGROUND: Coronary heart disease is currently the leading cause of death in humans. Its poor prognosis and high mortality are associated with myocardial ischemia, which leads to metabolic disorder-related cardiomyocyte apoptosis and reactive oxygen species (ROS) production. Previous cardiovascular metabolomics studies in humans and mice have shown that proline metabolism is severely altered after cardiomyocyte hypoxia. Proline dehydrogenase (PRODH) is located on the inner mitochondrial membrane and is an enzyme that catalyzes the first step of proline catabolism, which plays an important role in improving the cellular redox state. In vitro oxygen-glucose deprivation can mimic in vivo myocardial ischemic injury. This study is aimed at investigating whether enhancing proline metabolism by overexpressing PRODH can ameliorate hypoxia-induced injury in cardiomyocytes and to reveal the related altered metabolites and mechanistic pathway via untargeted metabolomics analysis. METHODS AND RESULTS: First, through public database analysis and RT-qPCR and western blot analyses in a cardiomyocyte hypoxia model, we found that the expression of the proline-degrading enzyme PRODH was downregulated after myocardial infarction and hypoxia exposure. Second, LDH assays, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), DHE staining, flow cytometric apoptosis analysis with DCFH and Annexin V-FITC/PI, and western blot analysis were used to assess the injury level in cardiomyocytes. Enhanced proline metabolism induced by PRODH overexpression reduced the levels of reactive oxidative stress and apoptosis, whereas PRODH knockdown had the opposite effects. Third, untargeted metabolomics analysis revealed that the protective effect was associated with significant changes in metabolism linked to sphingolipid signaling pathways, unsaturated fatty acid biosynthesis, phosphocreatine, glutathione disulfide, aminoacyl-tRNA biosynthesis, and ABC transporters. CONCLUSIONS: Our study demonstrated a protective effect of enhanced proline metabolism in cardiomyocytes under hypoxia, providing a novel strategy for exploring new treatments for coronary heart disease.

Autophagy is induced in human keratinocytes during human papillomavirus 11 pseudovirion entry.

Human papillomavirus type 11 (HPV11) is one of the main causes of condyloma acuminatum, a widespread sexually transmitted disease. During infection of its primary target cell, keratinocytes, it is likely to encounter the autophagy pathway, which is an intracellular maintenance process that is also able to target invading pathogens. It is currently unknown whether HPV11 is targeted by autophagy or whether it is able to escape autophagy-mediated killing. Here, we investigated the autophagy response during HPV11 pseudovirion (PsV) entry in human keratinocytes. Transmission electron microscopy showed that intracellular PsVs were sequestered in lumen of double-membrane autophagosomes that subsequently appeared to fuse with lysosomes, while confocal microscopy showed induction LC3 puncta, the hallmark of induced autophagy activity. Furthermore, quantitative infection assays showed that high autophagy activity resulted in reduced HPV11 PsV infectivity. Therefore, the autophagy pathway seemed to actively target invading HPV11 PsVs for destruction in the autolysosome. Western analysis on the phosphorylation state of autophagy regulators and upstream pathways indicated that autophagy was activated through interplay between Erk and Akt signaling. In conclusion, autophagy functions as a cellular protection mechanism against intracellular HPV11 and therefore therapies that stimulate autophagy may prevent recurrent condyloma acuminatum by helping eliminate latent HPV11 infections.

HPV E7 inhibits cell pyroptosis by promoting TRIM21-mediated degradation and ubiquitination of the IFI16 inflammasome.

Human papillomavirus (HPV) is a DNA virus that causes sexually transmitted infections. The HPV oncoprotein E7 plays a critical role in the regulation of host immunity to promote the immune escape of HPV and the occurrence of cervical cancer or genital warts. Pyroptosis, a highly inflammatory form of programmed cell death, can be induced by inflammasomes and acts as a defense against pathogenic infection. However, whether HPV E7 can regulate cell pyroptosis to evade immune surveillance has not been determined. In this study, we found that HPV E7 could inhibit cell pyroptosis induced by transfection with dsDNA. The activation of the inflammasome, and the production of IL-18 and IL-1ß were also restrained by HPV E7. Mass spectrometry and immunoprecipitation showed that HPV E7 interacted with IFI16 and TRIM21. We also discovered that HPV E7 recruited the E3 ligase TRIM21 to ubiquitinate and degrade the IFI16 inflammasome, leading to the inhibition of cell pyroptosis and self-escape from immune surveillance. Thus, our study reveals an important immune escape mechanism in HPV infection and may provide targets for the development of a novel immunotherapeutic strategy to effectively restore antiviral immunity.

Digital RNA Sequencing of Human Epidermal Keratinocytes Carrying Human Papillomavirus Type 16 E7.

High-risk human papillomavirus (HPV) infections are the predominant cause of cervical cancer and its early gene E7 plays an important role in cellular proliferation and cell-cycle progression. While tremendous progress has been made in exploring the molecular mechanisms in late tumorigenesis, many pathways showing how HPV deregulates host gene expression in early inapparent infections and early tumorigenesis still remain undefined. Digital RNA sequencing was performed and a total of 195 differentially expressed genes were identified between the HPV16 E7-transfected NHEKs and control cells (p < 0.05, fold-change > 2). GO enrichment showed that HPV16 E7 primarily affected processes involved in anti-viral and immune responses, while KEGG pathway analysis showed enrichment of gene clusters of associated with HPV infection and MAPK signaling. Of the differentially expressed genes, IFI6, SLC39A9 and ZNF185 showed a strong correlation with tumor progression and patient survival in the OncoLnc database while roles for AKAP12 and DUSP5 in carcinogenesis and poor prognosis have previously been established for other cancer types. Our study identified several novel HPV16 E7-regulated candidate genes with putative functions in tumorigenesis, thus providing new insights into HPV persistence in keratinocytes and early onset of tumorigenesis.

Proline improves cardiac remodeling following myocardial infarction and attenuates cardiomyocyte apoptosis via redox regulation.

At present, ischemic heart failure (HF) caused by coronary heart disease (CHD) has a high morbidity and mortality, placing a heavy burden on global human health. L-Proline (Pro), a nonessential amino acid and the foundation of proteins in the human body, was found to be protective against oxidative stress in various diseases. However, the role of Pro in cardiovascular disease (CVD) remains unclear. In vivo, adult mice were subjected to left anterior descending (LAD) artery ligation for 4 weeks with or without Pro treatment. In vitro, H9c2 cardiomyocytes were pretreated with or without Pro, followed by treatment with hydrogen peroxide (H2O2) (200 µM) for 6 and 12 h. Our data showed that Pro metabolism was disturbing after myocardial infarction (MI). Pro treatment improved cardiac remodeling, reduced infarct size, and decreased oxidative stress and apoptosis in mouse hearts after MI. Pro inhibited the H2O2-induced increase in reactive oxygen species (ROS) in H9c2 cells and protected against H2O2-induced apoptosis. Mechanistically, by RNA sequencing (RNA-seq) and pathway analysis, Pro was shown to exert a protective effect through H2O2 catabolic processes and apoptotic processes, especially oxidative phosphorylation (OXPHOS). Taken together, our findings suggested that Pro protects against MI injury at least partially via redox regulation, highlighting the potential of Pro as a novel therapy for ischemic HF caused by CHD.

Human Papillomavirus 11 Early Protein E6 Activates Autophagy by Repressing AKT/mTOR and Erk/mTOR.

Low-risk human papillomaviruses (LR-HPVs) are the causative agents of genital warts, which are a widespread sexually transmitted disease. How LR-HPVs affect autophagy and the specific proteins involved are unknown. In the current study, we investigated the impact of LR-HPV11 early protein 6 (E6) on the activity of the autophagy pathway. We transfected an HPV11 E6 (11E6) plasmid into HaCaT cells, H8 cells, and NHEK cells and established a stable cell line expressing the HPV11 E6 protein. The differences in autophagy activity and upstream regulatory pathways compared with those in the parent cell lines were investigated using a Western blot analysis of the total and phosphorylated protein levels and confocal microscopy of immunostained cells and cells transfected with an mCherry-green fluorescent protein-LC3 expression plasmid. We used short hairpin RNA (shRNA) to knock down 11E6 and showed that these effects require continued 11E6 expression. Compared with its expression in the control cells, the expression of HPV11 E6 in the cells activated the autophagy pathway. The increased autophagy activity was the result of the decreased phosphorylation levels of the canonical autophagy repressor mammalian target of rapamycin (mTOR) at its Ser2448 position (the mTOR complex 1 [mTORC1] phosphorylation site) and decreased AKT and Erk phosphorylation. Therefore, these results indicate that HPV11 E6 activates autophagy through the AKT/mTOR and Erk/mTOR pathways. Our findings provide novel insight into the relationship between LR-HPV infections and autophagy and could help elucidate the pathogenic mechanisms of LR-HPV.IMPORTANCE We transfected an HPV11 E6 plasmid into HaCaT cells, H8 cells, and NHEK cells and established a stable cell line expressing the HPV11 E6 protein. Then, we confirmed that HPV11 E6 induces autophagy by suppressing the AKT/mTOR and Erk/mTOR pathways. In contrast to the high-risk HPV E6 genes, HPV11 E6 did not affect the expression of p53. To the best of our knowledge, this study represents the first direct in-depth investigation of the relationship between the LR-HPV E6 gene and autophagy, which may help to reveal the pathogenesis of LR-HPV infection.